In the United States, 25.8 million children and adults have diabetes and 79 million have pre-diabetes. Insulin therapy is critical in Type 1 Diabetes and now understood to be an inevitable therapeutic component for Type 2 Diabetes in order to achieve adequate glycemic control. However, none of the available commercial insulin preparations are able to simulate normal secretion. Continuous glucose monitoring and insulin pumps have enabled progress toward maintaining euglycemia. However, they cannot account for the insulin absorption variability within and between individuals, which can result in hypoglycemia and hyperinsulinemia. Real-time insulin measurement could overcome this limitation. However, despite extensive research and advancements in the design of glucose sensors, the development of an analogous insulin monitor has yet to be achieved. This is due to three critical performance requirements that an ideal insulin sensor must meet: (1) resolve rising and falling insulin concentrations at physiological time-scales and concentrations; (2) operate continuously, without batch processing or exogenous reagents (3) retain a stable quantitative signal through prolonged exposure to unprocessed biological fluids. We propose to unlock the first real-time insulin sensor, by creating a disposable microfluidic device, which overcomes the critical performance challenges. In Phase I we focus on a proof of concept device to demonstrate real-time measurement of insulin doped into human whole blood in vitro. Our aims include: (1) Create an aptamer-based electrochemical insulin probe for measurement in buffer. Here we will develop a self-reporting probe that responds to dynamic insulin concentrations in buffer. (2) Create a continuous flow diffusion filter to support sensing from complex media. We will develop a microfluidic diffusion filter to selectively permits the passage of high-diffusivity insulin from whole blood to the sensor surface while rejecting low-diffusivity proteins and cells. (3) Develop stabilized quantitative output via auto-correcting differential measurement. Here we will exploit the frequency dependence of our probe to enable a new form of differential measurement, which eliminates signal drift for sustained insulin quantification in whole blood. We have preliminary data supporting each aim and we anticipate achieving accurate measurement of doped insulin in whole blood for 10 hours. In Phase II, we will enhance the sensor performance to detect in vivo insulin levels by discovering pM affinity insulin aptamers. We will miniaturize and integrate the device with micropumps for subcutaneous measurement. In collaboration with the Sansum Diabetes Research Institute, we will perform human tests and compare real-time output to reference lab insulin assays. This work leverages our unique experience in microfluidic device development, aptamer-based real-time monitoring, and high-performance aptamer discovery, and the vast clinical expertise of our Phase II collaborators. We expect to form a product co- development partnership with leaders in the diabetes care device market for commercialization within five years.